Gram-negative bacteria have an elaborate envelope that is composed of an outer membrane, the cell wall, and the inner membrane, which collectively encase the cytoplasm. The outer membrane is covalently anchored to the peptidoglycan, the major constituent of the cell wall. We document that the gene product of PA2854 is the catalyst that performs this transformation between the cell wall and the outer-membrane lipoprotein OprI in live Pseudomonas aeruginosa. Furthermore, we reconstitute this reaction in vitro with the use of purified recombinant PA2854, OprI, and synthetic samples of the cell-wall peptidoglycan, in each case documenting the attachment of the side-chain ε-amino group of Lys83 of OprI to the peptide stem of the peptidoglycan. OprI forms a trimeric structure. The X-ray structure of the trimeric state of OprI was solved to 2.1 Å resolution, which reveals it as an extended 82 Å helix bundle. The X-ray structure of PA2854 was also solved at 2.63 Å resolution. The enzyme is composed of three domains, which were documented to bind to the synthetic peptidoglycan and to OprI. The enzyme turns over both non-cross-linked and cross-linked peptidoglycan as its substrate. A model for the ternary assembly of the complex of PA2854-OprI-peptidoglycan is proposed based on the collective evidence. We document that anchoring of the outer membrane to the cell wall catalyzed by PA2854 does not appear to be a redundant reaction (product of more than one enzyme), in the absence of which the bacterium shows a weakened envelope, prone to disruption.

Spore germination in Clostridioides difficile is initiated by a cascade of activities of several proteins that culminates in the activation of SleC, a cell-wall-processing enzyme. We report herein the details of the enzymatic activities of SleC by the use of synthetic peptidoglycan fragments and of spore sacculi. The reactions include the formation of 1,6-anhydromuramate─a hallmark of lytic transglycosylase activity─as well as a muramate hydrolytic product, both of which proceed through the same transient oxocarbenium species. Furthermore, we report the first X-ray structure of zymogenic prepro-SleC at 2.1 Å resolution. Additionally, the structure provides insights into the YabG and CspB cleavage sites necessary for the activation of the zymogen. The active site of SleC presents relevant differences in contrast to SpoIID, a homologous lytic transglycosylase involved in the sporulation Clostridioides species, explaining the ability of SleC to turn over the spore sacculus, a prerequisite for the germination event. A screening of an in-house library of compounds led to the discovery of an oxadiazole that binds to the mature (activated) form of SleC, whereby it shuts down the ability of spores to germinate in the presence of germinants. This is consistent with the SleC activity as an end-point for the germination cascade. The mechanistic knowledge and the inhibitor hold the promise in addressing an unmet medical need in intervention of recurrent infections by C. difficile.

Pseudomonas aeruginosa is an opportunistic pathogen that frequently resides in multispecies communities. During chronic infections, P. aeruginosa employs a diverse arsenal of antibacterial weapons to compete with other bacteria for resources and space. Using genetic and biochemical approaches, we identified a type VI secretion system-dependent antibacterial effector-immunity pair, PseM (P. aeruginosa secreted endolytic muramidase) and PA0990 in P. aeruginosa. Our findings demonstrate that PseM functions as an endolytic muramidase, targeting prey bacteria by hydrolytically cleaving cell-wall peptidoglycan, whereas its immunity partner PA0990 provides self-protection. The X-ray crystal structure of PseM reveals a homodimeric configuration, with its active site formed by segments from both monomers. Through structural analysis and macromolecular docking simulations, we further elucidate the substrate-binding residues critical for the activity of PseM. Importantly, we show that PseM contributes to P. aeruginosa growth among bacterial competition. Together, these results uncover a novel antibacterial mechanism mediated by PseM, highlighting the dynamic nature of interspecies and intraspecies competition within bacterial populations.

Remodeling of the pneumococcal cell wall, carried out by peptidoglycan (PG) hydrolases, is imperative for maintaining bacterial cell shape and ensuring survival, particularly during cell division or stress response. The Streptococcus pneumoniae protein Spr1875 plays a role in stress response, both regulated by the VicRK two-component system (analogous to the WalRK TCS found in Firmicutes). Modular Spr1875 presents a putative cell-wall binding module at the N-terminus and a catalytic C-terminal module (Spr1875MT3) connected by a long linker. Assays of the full-length protein and Spr1875MT3 with PG-based synthetic substrates by liquid chromatography/mass spectrometry revealed Spr1875 as an l,d-endopeptidase, renamed VldE (for VicRK-regulated l,d-endopeptidase), which hydrolyzed the cross-linked stem peptide in the PG. Remarkably, we observed asymmetric turnover with specific recognition of the acceptor peptide strand. Localization experiments showed that the protein is directed to the septum, which suggests that muralytic activity could be required for pneumococcal growth under stress conditions. Our findings, based on six high-resolution X-ray crystallographic structures and molecular-dynamics simulations, reveal two states for VldEMT3. The protein transitions between a noncatalytic state that binds up to four zinc ions, thus behaving as a Zn2+ reservoir, and a catalytic state that performs the hydrolytic reaction with a single zinc ion. Furthermore, computational studies provide insight into the mechanism of catalytic-water activation and nucleophilic attack on the specific scissile peptide bond of the asymmetric cross-linked PG.

Bacterial cell-wall hydrolases must be tightly regulated during bacterial cell division to prevent aberrant cell lysis and to allow final separation of viable daughter cells. In a multidisciplinary work, we disclose the molec-ular dialogue between the cell-wall hydrolase LytB, wall teichoic acids, and the eukaryotic-like protein kinase StkP in Streptococcus pneumoniae. After characterizing the peptidoglycan recognition mode by the catalytic domain of LytB, we further demonstrate that LytB possesses a modular organization allowing the specific binding to wall teichoic acids and to the protein kinase StkP. Structural and cellular studies notably reveal that the temporal and spatial localization of LytB is governed by the interaction between specific modules of LytB and the final PASTA domain of StkP. Our data collectively provide a comprehensive understanding of how LytB performs final separation of daughter cells and highlights the regulatory role of eukaryotic-like kinases on lytic machineries in the last step of cell division in streptococci.

Clostridioides difficile is a spore-forming human pathogen responsible for significant morbidity and mortality. Infections by this pathogen ensue dysbiosis of the intestinal tract, which leads to germination of the spores. The process of spore formation requires a transition for the cell-wall peptidoglycan of the vegetative C. difficile to that of spores, which entails the formation of muramyl-δ-lactam. We describe a set of reactions for three recombinant C. difficile proteins, GerS, CwlD, and PdaA1, with the use of four synthetic peptidoglycan analogs. CwlD and PdaA1 excise the peptidoglycan stem peptide and the acetyl moiety of N-acetyl muramate, respectively. The reaction of CwlD is accelerated in the presence of GerS. With the use of a suitable substrate, we document that PdaA1 catalyzes a novel zinc-dependent transamidation/transpeptidation reaction, an unusual reaction that requires excision of the stem peptide as a pre-requisite.

The soluble lytic transglycosylase Cj0843c from Campylobacter jejuni breaks down cell-wall peptidoglycan (PG). Its nonhydrolytic activity sustains cell-wall remodeling and repair. We report herein our structure-function studies probing the substrate preferences and recognition by this enzyme. Our studies show that Cj0843c exhibits both exolytic and endolytic activities and forms the N-acetyl-1,6-anhydromuramyl (anhMurNAc) peptidoglycan termini, the typical transformation catalyzed by lytic transglycosylase. Cj0843c shows a trend toward a preference for substrates with anhMurNAc ends and those with peptide stems. Mutagenesis revealed that the catalytic E390 is critical for activity. In addition, mutagenesis showed that R388 and K505, located in the positively charged pocket near E390, also serve important roles. Mutation of R326, on the opposite side of this positively charged pocket, enhanced activity. Our data point to different roles for positively charged residues in this pocket for productive binding of the predominantly negatively charged PG. We also show by X-ray crystallography and by molecular dynamics simulations that the active site of Cj0843c is still capable of binding GlcNAc containing di- and trisaccharides without MurNAc moieties, without peptide stems, and without the anhMurNAc ends.

During evolution, enzymes can undergo shifts in preferred substrates or in catalytic activities. An intriguing question is how enzyme function changes following horizontal gene transfer, especially for bacterial genes that have moved to animal genomes. Some insects have acquired genes that encode enzymes for the bio-synthesis of bacterial cell wall components and that appear to function to support or control their obligate endosymbiotic bacteria. In aphids, the bacterial endosymbiont Buchnera aphidicola provides essential amino acids for aphid hosts but lacks most genes for remodeling of the bacterial cell wall. The aphid genome has acquired seven genes with putative functions in cell wall metabolism that are primarily expressed in the aphid cells harboring Buchnera. In analyses of aphid homogenates, we detected peptidoglycan (PGN) muropeptides indicative of the reactions of PGN hydrolases encoded by horizontally acquired aphid genes but not by Buchnera genes. We produced one such host enzyme, ApLdcA, and characterized its activity with both cell wall derived and synthetic PGN. Both ApLdcA and the homologous enzyme in Escherichia coli, which functions as an L,D-carboxypeptidase in the cytoplasmic PGN recycling pathway, exhibit turnover of PGN substrates containing stem pentapeptides and cross-linkages via L,D-endopeptidase activity, consistent with a potential role in cell wall remodeling. Our results suggest that ApLdcA derives its functions from the promiscuous activities of an ancestral LdcA enzyme, whose acquisition by the aphid genome may have enabled hosts to influence Buchnera cell wall metabolism as a means to control symbiont growth and division. IMPORTANCE Most enzymes are capable of performing biologically irrelevant side reactions. During evolution, promiscuous enzyme activities may acquire new biological roles, especially after horizontal gene transfer to new organisms. Pea aphids harbor obligate bacterial symbionts called Buchnera and encode horizontally acquired bacterial genes with putative roles in cell wall metabolism. Though Buchnera lacks cell wall endopeptidase genes, we found evidence of endopeptidase activity among peptidoglycan muropeptides purified from aphids. We characterized a multifunctional, aphid-encoded enzyme, ApLdcA, which displays L,D-endopeptidase activities considered promiscuous for the Escherichia coli homolog, for which these activities do not contribute to its native role in peptidoglycan recycling. These results exemplify the roles of enzyme promiscuity and horizontal gene transfer in enzyme evolution and demonstrate how aphids influence symbiont cell wall metabolism.

The penicillin-binding proteins are the enzyme catalysts of the critical transpeptidation crosslinking polymerization reaction of bacterial peptidoglycan synthesis and the molecular targets of the penicillin antibiotics. Here, we report a combined crystallographic, small-angle X-ray scattering (SAXS) in-solution structure, computational and biophysical analysis of PBP1 of Staphylococcus aureus (saPBP1), providing mechanistic clues about its function and regulation during cell division. The structure reveals the pedestal domain, the transpeptidase domain, and most of the linker connecting to the "penicillin-binding protein and serine/threonine kinase associated" (PASTA) domains, but not its two PASTA domains, despite their presence in the construct. To address this absence, the structure of the PASTA domains was determined at 1.5 Å resolution. Extensive molecular-dynamics simulations interpret the PASTA domains of saPBP1 as conformationally mobile and separated from the transpeptidase domain. This conclusion was confirmed by SAXS experiments on the full-length protein in solution. A series of crystallographic complexes with β-lactam antibiotics (as inhibitors) and penta-Gly (as a substrate mimetic) allowed the molecular characterization of both inhibition by antibiotics and binding for the donor and acceptor peptidoglycan strands. Mass-spectrometry experiments with synthetic peptidoglycan fragments revealed binding by PASTA domains in coordination with the remaining domains. The observed mobility of the PASTA domain in saPBP1 could play a crucial role for in vivo interaction with its glycosyltransferase partner in the membrane or with other components of the divisome machinery, as well as for coordination of transpeptidation and polymerization processes in the bacterial divisome.

Assembly of the peptidoglycan is crucial in maintaining viability of bacteria and in defining bacterial cell shapes, both of which are important for existence in the ecological niche that the organism occupies. Here, eight crystal structures for a member of the cell-shape-determining class of Campylobacter jejuni , the peptidoglycan peptidase 3 (Pgp3), are reported. Characterization of the turnover chemistry of Pgp3 reveals cell wall d , d -endopeptidase and d , d -carboxypeptidase activities. Catalysis is accompanied by large conformational changes upon peptidoglycan binding, whereby a loop regulates access to the active site. Furthermore, prior hydrolysis of the crosslinked peptide stem from the saccharide backbone of the peptidoglycan on one side is a pre-requisite for its recognition and turnover by Pgp3. These analyses reveal the noncanonical nature of the transformations at the core of the events that define the morphological shape for C. jejuni as an intestinal pathogen.

Diabetic foot ulcers (DFUs) are a common complication of diabetes that are recalcitrant to healing due to persistent inflammation. The majority of DFUs have bacterial biofilms, with Staphylococcus epidermidis as a predominant bacterium, requiring infection control with antibiotics before treatment of the wound. Matrix metalloproteinases (MMPs) play roles in the pathology and repair of DFUs. However, defining the roles of the 24 human MMPs has been challenging due to the presence of three forms for each MMP, of which only one is catalytically competent, and the lack of convenient methods to distinguish among the three forms of MMPs. Using an affinity resin that binds only to the active forms of MMPs, with identification and quantification by mass spectrometry, we found that infected wounds in mice had increased levels of active MMP-9 compared to uninfected ones, paralleling infected human DFUs. MMP-9 activity prevents diabetic wounds from healing. We evaluated the efficacy of the selective small-molecule MMP-9 inhibitor, (R)-ND-336, in the infected diabetic mouse model of wound healing and showed that (R)-ND-336 alone or in combination with the antibiotic linezolid improves wound healing by inhibiting the detrimental MMP-9, mitigating macrophage infiltration to diminish inflammation, and increasing angiogenesis to restore the normal wound healing process. An advantage of this strategy is the ability to administer (R)-ND-336 concurrently with an antibiotic.

BglX is a heretofore uncharacterized periplasmic glycoside hydrolase (GH) of the human pathogen Pseudomonas aeruginosa. X-ray analysis identifies it as a protein homodimer. The two active sites of the homodimer comprise catalytic residues provided by each monomer. This arrangement is seen in <2% of the hydrolases of known structure. In vitro substrate profiling shows BglX is a catalyst for beta-(1 -> 2) and beta-(1 -> 3) saccharide hydrolysis. Saccharides with beta-(1 -> 4) or beta-(1 -> 6) bonds, and the beta-(1 -> 4) muropeptides from the cell-wall peptidoglycan, are not substrates. Additional structural insights from X-ray analysis (including structures of a mutant enzyme-derived Michaelis complex, two transition-state mimetics, and two enzyme product complexes) enabled the comprehensive description of BglX catalysis. The half-chair (H-4(3)) conformation of the transition-state oxocarbenium species, the approach of the hydrolytic water molecule to the oxocarbenium species, and the stepwise release of the two reaction products were also visualized. The substrate pattern for BglX aligns with the [beta-(1 -> 2)-Glc](x) and [beta-(1 -> 3)-Glc](x) periplasmic osmoregulated periplasmic glucans, and possibly with the Psl exopolysaccharides, of P. aeruginosa. Both polysaccharides are implicated in biofilm formation. Accordingly, we show that inactivation of the bglX gene of P. aeruginosa PAO1 attenuates biofilm formation.

The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell wall-derived muropeptides. AmpD is cytoplasmic and plays a role in the recycling of cell wall muropeptides, with a link to antibiotic resistance. AmpDh2 is a periplasmic soluble enzyme with the former anchored to the inner leaflet of the outer membrane. We document, herein, that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa delivers AmpDh3 (but not AmpD or AmpDh2) to the periplasm of a prey bacterium upon contact. AmpDh3 hydrolyzes the cell wall peptidoglycan of the prey bacterium, which leads to its killing, thereby providing a growth advantage for P. aeruginosa in bacterial competition. We also document that the periplasmic protein PA0808, heretofore of unknown function, affords self-protection from lysis by AmpDh3. Cognates of the AmpDh3-PA0808 pair are widely distributed across Gram-negative bacteria. Taken together, these findings underscore the importance of their function as an evolutionary advantage and that of the H2-T6SS as the means for the manifestation of the effect.

SPOR domains are widely present in bacterial proteins that recognize cell-wall peptidoglycan strands stripped of the peptide stems. This type of peptidoglycan is enriched in the septal ring as a product of catalysis by cell-wall amidases that participate in the separation of daughter cells during cell division. Here, we document binding of synthetic denuded glycan ligands to the SPOR domain of the lytic transglycosylase RlpA from Pseudomonas aeruginosa (SPOR-RlpA) by mass spectrometry and structural analyses, and demonstrate that indeed the presence of peptide stems in the peptidoglycan abrogates binding. The crystal structures of the SPOR domain, in the apo state and in complex with different synthetic glycan ligands, provide insights into the molecular basis for recognition and delineate a conserved pattern in other SPOR domains. The biological and structural observations presented here are followed up by molecular-dynamics simulations and by exploration of the effect on binding of distinct peptidoglycan modifications.

Diabetic foot ulcers are characterized by hypoxia. For many patients, hyperbaric oxygen (HBO) therapy is the last recourse for saving the limb from amputation, for which the molecular basis is not understood. We previously identified the active form of matrix metalloproteinase-9 (MMP-9) as responsible for diabetic foot ulcer's recalcitrance to healing. Transcription of mmp-9 to the inactive zymogen is upregulated during hypoxia. Activation of the zymogen is promoted by proteases and reactive oxygen species (ROS). We hypothesized that the dynamics of these two events might lead to a lowering of active MMP-9 levels in the wounded tissue. We employed the full-thickness excisional db/db mouse model to study wound healing, and treated the mice to 3.0 atm of molecular oxygen for 90 minutes, 5 days per week for 10 days in an HBO research chamber. Treatment with HBO accelerated diabetic wound healing compared to untreated mice, with more completed and extended reepithelialization. We imaged the wounds for ROS in vivo with a luminol-based probe and found that HBO treatment actually decreases ROS levels. The levels of superoxide dismutase, catalase, and glutathione peroxidase-enzymes that turn over ROS-increased after HBO treatment, hence the observation of decreased ROS. Since ROS levels are lowered, we explored the effect that this would have on activation of MMP-9. Quantitative analysis with an affinity resin that binds and pulls down the active MMPs exclusively, coupled with proteomics, revealed that HBO treatment indeed reduces the active MMP-9 levels. This work for the first time demonstrates that diminution of active MMP-9 is a contributing factor and a mechanism for enhancement of diabetic wound healing by HBO therapy.

The human pathogen Pseudomonas aeruginosa harbors three paralogous zinc proteases annotated as AmpD, AmpDh2, and AmpDh3, which turn over the cell wall and cell-wall-derived muropeptides. AmpD is cytoplasmic and plays a role in recycling of cell-wall muropeptides, with a link to antibiotic resistance. AmpDh2 and AmpDh3 are periplasmic enzymes; with the former anchored to the inner leaflet of the outer membrane and the latter a soluble enzyme. We document herein that the type VI secretion system locus II (H2-T6SS) of P. aeruginosa delivers AmpDh3 (but not AmpD or AmpDh2) to the periplasm of a prey bacterium upon contact. AmpDh3 hydrolyzes the cell-wall peptidoglycan of the prey bacterium, which leads to its killing, thereby providing a growth advantage for P. aerugionsa in bacterial competition. We also document that the periplasmic protein PA0808, heretofore of unknown function, affords self-protection from lysis by AmpDh3. Cognates of the AmpDh3-PA0808 pair are widely distributed across Gram-negative bacteria, underscoring the importance of their function as an evolutionary advantage and that of the H2-T6SS as the means for manifestation of the effect.

Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan, and a noncovalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A prerequisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall cross-linking reaction.

Farnesol, a sesquiterpene alcohol, potentiates the activity of β-lactam antibiotics against antibiotic-resistant bacteria. We document that farnesol and two synthetic derivatives (compounds 2 and 6) have poor antibacterial activities of their own, but they potentiate the activities of ampicillin and oxacillin against Staphylococcus aureus strains (including methicillin-resistant S. aureus). These compounds attenuate the rate of growth of bacteria, which has to be taken into account in assessment of the potentiation effect.

Diabetic foot ulcers (DFUs) are a significant health problem. A single existing FDA-approved drug for this ailment, becaplermin, is not standard-of-care. We previously demonstrated that upregulation of active matrix metalloproteinase (MMP)-9 is the reason that the diabetic wound in mice is recalcitrant to healing and that MMP-8 participates in wound repair. In the present study, we validate the target MMP-9 by identifying and quantifying active MMP-8 and MMP-9 in human diabetic wounds using an affinity resin that binds exclusively to the active forms of MMPs coupled with proteomics. Furthermore, we synthesize and evaluate enantiomerically pure ( R)- and ( S)-ND-336, as inhibitors of the detrimental MMP-9, and show that the ( R)-enantiomer has superior efficacy in wound healing over becaplermin. Our results reveal that the mechanisms of pathology and repair are similar in diabetic mice and diabetic humans and that ( R)-ND-336 holds promise for the treatment of DFUs as a first-in-class therapeutic.